Objective To explore the effects of enterovirus-A71 (EV-A71) structural and non-structural proteins on expression of p85 and activation of p38 pathway. Methods Twelve gene segments of EV-A71 were amplified by RT-PCR. The target fragments were ligated into the pcDNA3.1(+)/myc-His A eukaryotic expression vector after double digestion by restriction enzymes and ligation reaction, to prepare the endotoxin-free recombinant plasmid. Twelve recombinant plasmids were transfected into HEK293 cells using SuperFect transfection reagent for 24h after endotoxin-free plasmids were prepared. The dose of each plasmid per well was 2μg. Expression of p85 and phosphorylation level of p38 were detected through western blotting. Results Twelve recombinant eukaryotic expression plasmids of EV-A71 structural and non-structural proteins were constructed successfully. Compared with untransfected cells, the expression of p85 and the phosphorylation level of p38 were regulated by EV-A71 structural proteins in P1 region (VP1, VP3 and VP4), non-structural proteins in P2 region (2A, 2B, 2C and 2BC) and non-structural proteins in P3 region (3A, 3C, 3D and 3AB) in different degrees. Although EV-A71 structural protein VP2 could not significantly elevate the expression of p85, the phosphorylation level of p38 was increased. Conclusion Twelve subunit proteins of EV-A71 interact with host cells, which further regulate the biological process of host cells.